The growing field of proteomics is concerned with identifying large numbers of proteins in living organisms, understanding the functions and interactions of these proteins, and characterizing how the repertoire of proteins in an organism is modulated by factors such as developmental stage, disease state, and environment. Proteomics also aims to address how the set of proteins needed to sustain life varies among individual cells, cell types, tissues, individual organisms, groups of organisms, species, and groups of species. An important tool in experimental proteomics is mass spectroscopy, which allows identification of many proteins from complex biological samples. In many mass spectroscopy protocols, proteins or protein fragments from a sample are ionized and detected on the basis of mass. The detected masses are then compared, through a database search, with the predicted masses of proteins (or portions thereof) thought to exist in the organism from which the sample is obtained. Matches between detected and predicted masses allow the amino acid sequences of the particular proteins present in the sample to be inferred.
Crude biological samples that contain diverse populations of proteins are often not pure enough to submit directly to mass spectroscopy. These samples can also contain carbohydrates, lipids, nucleic acids, and other contaminants, which, if not removed prior to data acquisition, can lead to artefactual peaks on a mass spectrum and masking of the peaks arising from proteins of interest. Furthermore, proteins cannot be easily identified by mass spectroscopy if too many different proteins are passed through the mass spectrometer at once. This can lead to highly complex mass spectra where individual peaks, and in turn, individual protein sequences cannot be easily resolved. Accordingly, biological samples often must be processed for proteomic mass spectroscopy, such that proteins are separated from contaminants and from each other before being injected into the mass spectrometer.
Gel electrophoresis and liquid chromatography (LC) are two techniques that are often used together for this purpose. Gel electrophoresis separates proteins on the basis of mass, size, or isoelectric point, while liquid chromatography can separate molecules on the basis of hydrophobicity, hydrophilicity, size, charge, affinity for a binding partner, and other characteristics (depending on the nature of the chromatography column). Using the two techniques in series allows proteins to separated from each other and from any contaminants on the basis of two or more orthogonal characteristics, so that they can be more easily analyzed and identified by mass spectroscopy. In practice, a protein sample is run on an electrophoresis gel, and the resulting separation allows extraction of specific portions of the sample from corresponding regions of the gel. The portions are then passed through a liquid chromatography column one by one and fed into a mass spectrometer, and multiple spectra can be acquired as the elution of proteins from the column progresses. The use of gel electrophoresis in conjunction with liquid chromatography and mass spectroscopy is referred to herein, and in the art, as GeLC-MS.
To extract proteins from a gel, the proteins must first be detected after electrophoresis. Detection tells the practitioner where proteins are located in the gel and allows him or her to identify specific portions of the protein sample. In the case of a two-dimensional (2D) gel, i.e. an electrophoresis gel used to separate proteins in two orthogonal directions on the basis of two different physical properties, detection reveals the locations of individual proteins or groups of proteins, which appear as spots. In the case of a one-dimensional (1D) gel, where separation occurs in only one direction, detection reveals the farthest extent that proteins have migrated from the wells in which they were loaded, the distribution of proteins along the direction of migration, and the boundaries of lanes. Here, proteins appear as bands or as a streak on the gel. Specific portions of the protein sample can then be chosen as desired by the practitioner for extraction, and the information provided by detection can be used subsequently, for example to interpret mass spectroscopy data.
Detection of proteins in electrophoresis gels is frequently performed using colored or fluorescent protein stains such as Coomassie Brilliant Blue or SYPRO Ruby. These stains can bind to proteins non-covalently, in a manner that is largely independent of amino acid sequence, and can be visualized upon illumination with specific wavelengths of light. Protein stains allow robust and sensitive detection, but hinder the rapid processing of biological samples for proteomic mass spectroscopy. The process of applying the stain to the gel (staining) prior to detection can take hours. Similarly time consuming is the removal of the stain after detection (destaining), which can be necessary in order to obtain accurate masses of proteins and protein fragments. Staining and destaining involve agitation of the gel, prolonged immersion of the gel in an aqueous buffer, and frequent changes of this buffer. During these processes, some proteins (particularly low-molecular-weight and hydrophilic proteins) can diffuse out of the gel and into the buffer, thereby becoming lost to subsequent analysis. Use of protein stains, and the accompanying gel handling, can therefore reduce the number of proteins that can be identified in GeLC-MS.